Function of the CysD domain of the gel-forming MUC2 mucin
| Autor: | Ute Krengel, Elisabeth Thomsson, Daniel Ambort, Gunnar C. Hansson, Jenny Mackenzie, Malin E. V. Johansson, Sjoerd van der Post |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2011 |
| Předmět: |
Glycosylation
CID collision-induced dissociation BN-PAGE blue native PAGE disulfide bonds Mucin 2 Biochemistry chemistry.chemical_compound Cricetinae CK domain cystine-knot domain MS/MS tandem MS Disulfides Peptide sequence 0303 health sciences ESI electrospray ionization AGC automatic gain control LC liquid chromatography Chemistry 030302 biochemistry & molecular biology Covalent bond non-covalent dimer Electrophoresis Polyacrylamide Gel Domain of unknown function Dimerization Research Article Stereochemistry Recombinant Fusion Proteins Molecular Sequence Data Size-exclusion chromatography CHO Cells TCEP-HCl tris(2-carboxyethyl)phosphine-HCl PTS domain proline threonine and serine domain 03 medical and health sciences Cricetulus C-mannosylation FBS fetal bovine serum mucus Animals Humans mass spectrometry (MS) Amino Acid Sequence mAb monoclonal antibody Molecular Biology 030304 developmental biology Mucin-2 EK enterokinase Mucin IMDM Iscove's modified Dulbecco's medium Cell Biology Fusion protein Protein Structure Tertiary CHO Chinese-hamster ovary |
| Zdroj: | Biochemical Journal; Vol 436 Biochemical Journal |
| ISSN: | 0264-6021 |
| DOI: | 10.1042/bj20102066 |
| Popis: | The colonic human MUC2 mucin forms a polymeric gel by covalent disulfide bonds in its N- and C-termini. The middle part of MUC2 is largely composed of two highly O-glycosylated mucin domains that are interrupted by a CysD domain of unknown function. We studied its function as recombinant proteins fused to a removable immunoglobulin Fc domain. Analysis of affinity-purified fusion proteins by native gel electrophoresis and gel filtration showed that they formed oligomeric complexes. Analysis of the individual isolated CysD parts showed that they formed dimers both when flanked by two MUC2 tandem repeats and without these. Cleavages of the two non-reduced CysD fusion proteins and analysis by MS revealed the localization of all five CysD disulfide bonds and that the predicted C-mannosylated site was not glycosylated. All disulfide bonds were within individual peptides showing that the domain was stabilized by intramolecular disulfide bonds and that CysD dimers were of non-covalent nature. These observations suggest that CysD domains act as non-covalent cross-links in the MUC2 gel, thereby determining the pore sizes of the mucus. |
| Databáze: | OpenAIRE |
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